Vertical plug-flow process for bio-conversion employing microorganisms

ABSTRACT

The invention relates to a method for producing a solid transformation product of a substrate comprising the following steps: •preparing a substrate of biomass comprising carbohydrates and proteinaceous matter that originates from soya bean, rape seed, or mixtures thereof, optionally in further mixture with carbohydrates and proteinaceous matter originating from fava beans, peas, sunflower seeds, lupine, cereals, and/or grasses, •mixing said substrate with a live microorganism or a combination of live microorganisms, which live microorganism or mixture of live microorganisms is not live yeast, and adding water in an amount which provides an initial incubation mixture having a water content from 30 to 70% by weight, and a ratio of wet bulk density to dry bulk density from 0.60 to 1.45 in the resulting mixture; •incubating said initial incubation mixture for 1-240 hours at a temperature of 15-70° C.; and thereafter recovering wet solid transformation product from the incubation mixture; further comprising that the incubating step is performed as a continuous plug-flow process in a vertical, non-agitated incubation tank with inlet means for said mixture and additives and outlet means for said solid transformation product.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is the U.S. National Stage of InternationalApplication No. PCT/EP2018/086282, filed Dec. 20, 2018, and claimspriority to European Patent Application No. 17210105.7 filed Dec. 22,2017.

FIELD OF THE INVENTION

The present invention relates to a solid substrate, bio-conversionmethod for the production of a valuable solid transformation product ofthe substrate wherein the bio-conversion is carried out by the use ofone or more suitable microorganism by a continuous plug flow process ina vertical, non-agitated tank where the transport is mediated bygravitational force.

BACKGROUND OF THE INVENTION

There is a need for bio-products that primarily can be used as food orfeed or as ingredients in food or feed. The basic constituents in suchproducts are proteins, fats, and carbohydrates. Suitable biomasses forsuch products are oil bearing crops such as oilseeds, cereals, andlegumes. Cereals have a protein content up to 15% e.g. in wheat, andlegumes have a protein content of up to 40% e.g. in soya beans, based ondry matter.

There is a similar need for the development of bio-products comprisingorganic compounds, such as organic acids, e.g. formic acid, acetic acid,propionic acid, butyric acid, lactic acid, and succinic acid, oralcohols, such as ethanol, which bio-products and organic compounds canbe produced in a cost-effective way in well-known processes usingmicroorganism genera which produce one or more organic compounds asmetabolic product of carbohydrate bio-conversion.

The lactic acid bacteria genera produce organic acid, in particularlactic acid and acetic acid, as their major metabolic end product ofcarbohydrate bio-conversion. The lactic acid bacteria genera are inparticular, but are not limited to, Lactobacillus, Leuconostoc,Pediococcus, Lactococcus, Enterococcus, Streptococcus, and Weisella.

Other microorganism genera also produce acids as their metabolic endproducts of carbohydrate bio-conversion. Such genera which produceorganic acid are in particular, but are not limited to, acid-producingBacillus, Bifidobacterium, Brevibacillus, Propionibacterium, Candida,Clostridium, and Geobacillus.

A general problem especially related to pulses and fruits and seeds fromlegumes as sources of bio-product and organic acids are the content ofindigestible oligosaccharides, such as stachyose and raffinose, causingflatulence and diarrhoea when fermented in the colon.

Low cost incubation methods known in the art are solid substrate orsolid-state fermentation (SSF) processes performed with low watercontent. The process consists of a solid, moist substrate inoculatedwith suitable microorganisms and left for bio-conversion undertemperature controlled conditions for a period of time.

Normally the substrate is incubated batch wise on flatbeds withoutstirring; one example of this process is known as the Koji process.Batch processes are also performed using stirring means.

Continuous SSF processes are also described in literature using thefollowing bioreactors: Stirred tank, rotating drum and tubular flowreactors. One example of a tubular flow reactor is the screw conveyortype.

U.S. Pat. No. 4,735,724 discloses a non-mixed vertical tower anaerobicdigester and a process for digestion of the biodegradable part offeedstock by methane producing microorganisms. The method ischaracterized in that there is a withdrawal of liquid from a middle orlower zone to the top of the tower.

EP 2 453 004 B1 discloses a method for anaerobic fermentation of organicmaterial in a closed tank and top down feed in the tank under the actionof the gravity. The method is characterized in that the fermenting massis agitated by alternately increasing the pressure of the product gasand abruptly relieving the pressure of the product gas.

The object of the present invention is to provide an improved method forthe production of a solid transformation product of a biomass substratein a vertical, plug flow, bio-conversion process carried out by the useof one or more suitable microorganism.

Another object is to provide a method, which can be performed in alarger but simpler reactor design than the prior art design.

Yet an object is to provide an efficient and fast method forbio-conversion of biomasses, in particular soya bean or rape seed ormixtures thereof, so as to produce bio-products comprising organiccompounds, such as organic acids, e.g. formic acid, acetic acid,propionic acid, butyric acid, lactic acid, and succinic acid, andalcohols, from cheap carbohydrate sources.

These objects are fulfilled with the method of the present invention.

SUMMARY OF THE INVENTION

Accordingly, in one aspect of the present invention it relates to amethod for producing a solid transformation product of a substratecomprising the following steps:

-   -   preparing a substrate of biomass comprising carbohydrates and        proteinaceous matter that originates from soya bean, rape seed,        or mixtures thereof, optionally in further mixture with        carbohydrates and proteinaceous matter originating from fava        beans, peas, sunflower seeds, lupine, cereals, and/or grasses,    -   mixing said substrate with a live microorganism or a combination        of live microorganisms, which live microorganism or combination        of live microorganisms is not live yeast, and adding water in an        amount which provides an initial incubation mixture having a        water content from 30% to 70%, and a ratio of wet bulk density        to dry bulk density from 0.60 to 1.45 in the resulting mixture;    -   incubating said initial incubation mixture for 1-240 hours at a        temperature of 15-70° C.; and recovering wet solid        transformation product from the incubated mixture;

further comprising that the incubating step is performed as a continuousplug-flow process in a vertical, non-agitated incubation tank with inletmeans for said mixture and additives and outlet means for said solidtransformation product.

The present method for treatment of biomass uses gravitational force totransport/move the biomass during incubation/bio-conversion. Althoughthe use of gravity for transportation in general is straightforward, itrequires careful selection of reaction conditions for the specificpurpose, such as in the case of the present plug-flow process.

Normally, when the water content is increased, an incubation mixturetends to compact, by the reduction of void volume, so that thetransportation behaviour is affected negatively. When a certain watercontent is reached the mixture is compacted to an extent so that thetransportation by gravitational force is stopped. The material may stickto the walls of the reactor, or it may create sedimentation, and theuniform plug-flow is disrupted resulting in uneven retention time of thebiomass.

Furthermore, if the bio-conversion is performed at elevated pressure,which may be the case under the gravity effect, the incubation reactiontends to slow down.

The solution according to the present invention to the problem connectedwith transportation by gravitational force of the incubation mixture isto make use of a tank as defined in the claims for incubation whereinthe flow of material can be kept so high and uniform that plug-flowconditions are achieved and maintained. The flow rate is regulated bythe inlet and outlet means and by the dimensions (width to height ratio)of the tank.

Furthermore, the solution according to the invention must securebalancing of the water content in the incubation mixture so that thewater activity on the particle surface is sufficient for the reactionprocess. This is achieved by keeping the ratio wet bulk density to drybulk density of the substrate low and within certain limits as definedin claim 1.

More specifically, the present inventors have found that the necessaryuniform process can be achieved by using an initial incubation mixturehaving a water content from 30% to 70% by weight, and a ratio of wetbulk density to dry bulk density from 0.60 to 1.45. In combination withthe present, vertical design for the plug-flow process it is possible tosecure a uniform plug-flow and ensure the same processing time for theincubation mixture. Furthermore, the method of the present invention isconducted without agitation. If the water content exceeds approximately70% by weight, the biomass cannot hold the water, and the incubationmixture becomes a slurry having a water phase and a solid phase. Thesetwo phases will not flow with the same flow rates, uniform plug flowwill not be obtained, and the incubation mixture may stick to theincubator walls. A water content of more than approximately 70% willresult in a ratio of wet bulk density to dry bulk density, exceeding1.45 that is the upper limit according to the invention.

The vertical design is less expensive in investment than a horizontaldesign due to its larger capacity in a single production line. It isalso less expensive to maintain due to less mechanical movements. Theuse of a non-agitated tank further contributes to reduced operationalcosts.

Thus, the present method allows an efficient and fast set-up of theprocess whereby the microorganism can propagate in liquid phase andperform bioconversion on cheap carbohydrate-based sources.

The present method is, in particular, efficient if the substrate ofbiomass has been pre-treated before it is mixed with the livemicroorganism or combination of live microorganisms, because thepre-treatment improves the access of the microorganisms to thecomponents in the biomass which are to be transformed. The pre-treatmentis typically carried out by chemical or physical pre-treatment, e.g. bymeans of disintegration, milling, flaking, heat treatment, pressuretreatment, ultrasonic treatment, hydrothermal treatment, or acid oralkaline treatment.

The method of the invention can be used to provide a solidtransformation product of the substrate which is a product of thetransformation of carbohydrates and/or proteins originating from saidbiomass. Such solid transformation products can be used e.g. in aprocessed food product or as an ingredient in a food or feed product oras an ingredient of a cosmetic or a pharmaceutical product, or anutritional supplement.

Definitions

In the context of the current invention, the following terms are meantto comprise the following, unless defined elsewhere in the description.

The terms “about”, “around”, “approximately”, or “˜” are meant toindicate e.g. the measuring uncertainty commonly experienced in the art,which can be in the order of magnitude of e.g. +/−1, 2, 5, 10, 20, oreven 50%.

The term “comprising” is to be interpreted as specifying the presence ofthe stated part(s), step(s), feature(s), composition(s), chemical(s), orcomponent(s), but does not exclude the presence of one or moreadditional parts, steps, features, compositions, chemicals orcomponents. E.g., a composition comprising a chemical compound may thuscomprise additional chemical compounds, etc.

Plug-Flow Process:

In this type of continuous process, the reaction mixture flows throughe.g. a tubular or polyhedral reactor with limited back mixing. The flowis a laminar flow where the composition of the reaction mixture changesalong the axial direction of the reactor, or a uniform mass flow.

Biomass:

Comprises biological material, as produced by the photosynthesis andthat can be used as raw material in industrial production. In thiscontext, biomass refers to plant matter in the form of seeds, cereals,pulses, grasses, e.g. beans and peas, etc., and mixtures thereof, and inparticular fruits and seeds of legumes. Furthermore, a biomasscomprising pulses is specifically applicable due to the protein contentand composition.

The substrate of biomass may be disintegrated by pre-treatment, such aschemical or physical pre-treatment, e.g. by means of disintegration,milling, flaking, heat treatment, pressure treatment, ultrasonictreatment, hydrothermal treatment, or acid or alkaline treatment.

Bio-Conversion/Incubation:

Is the process to incubate cultures of microorganisms on a substrate fora specific purpose, e.g. incubating a microorganism on a carbohydrate toproduce organic acids or alcohols.

Solid Transformation Product of the Substrate:

In general treatment of biomass by incubation with microorganisms can bedivided into four types:

-   -   Production of biomass—cellular material    -   Production of extracellular components—chemical compounds,        metabolites, such as acids, enzymes    -   Production of intracellular components—enzymes, etc.    -   Transformation product of the substrate—the transformed        substrate is the product

In the present context, solid transformation product of the substraterefers to a product resulting from incubation of the selected biomasswith live microorganism and optionally processing aids.

Bulk Density:

Bulk density is a parameter important for the physical behaviour of abiomass which has the form of powder, granules, and the like. Theparameter is defined as weight per volume, and may be measured in, e.g.,g/ml. It is not an intrinsic property, but can change depending onhandling, and can be used as an index of structural changes. The densityof a material is determined by placing a fixed volume of the material ina measuring cup and determining the weight or by determining the weightof a measured volume of a powder. By this test the following featurescan be determined:

Bulk density (also known as pour density)=mass/untapped dry volume ing/mL or kg/m³;

Wet bulk density (also known as total density)=the ratio of the totalmass (M_(s)+M_(l)) to its total volume;

M_(s)=mass of solids and M_(l)=mass of liquids.

Thus, in the context of the present invention, “dry bulk density” is themeasured bulk density of the biomass without addition of water, viz. thebulk density/pour density. “Wet bulk density” is the bulk densitymeasured after addition of a certain amount of water.

Normally, the bulk density is determined in accordance withInternational Standards ISO 697 and ISO 60, but due to the nature of thesubstances this was not applicable in the present context. Theindividual method used is described in the examples.

Oligosaccharides and Polysaccharides:

An oligosaccharide is a saccharide polymer containing at least twocomponent monomer sugars. Polysaccharides are saccharide polymerscontaining many component monomer sugars, also known as complexcarbohydrates. Examples include storage polysaccharides such as starchand structural polysaccharides such as cellulose.

Carbohydrates:

Comprise mono-, di-, oligo- and polysaccharides.

Proteinaceous Materials:

Comprise organic compounds with a substantial content of proteins madeof amino acids arranged in one or more chains. At a chain length of upto approximately 50 amino acids the compound is called a peptide; athigher molecular weight the organic compound is called a polypeptide ora protein.

Fats:

Comprise esters between fatty acids and glycerol. One molecule ofglycerol can be esterified to one, two and three fatty acid moleculesresulting in a monoglyceride, a diglyceride or a triglyceriderespectively. Usually fats consist of mainly triglycerides and minoramounts of lecithins, sterols, etc. If the fat is liquid at roomtemperature it is normally called oil. With respect to oils, fats, andrelated products in this context, reference is made to “Physical andChemical Characteristics of Oils, Fats and Waxes”, AOCS, 1996, as wellas “Lipid Glossary 2”, F. D. Gunstone, The Oily Press, 2004.

Glycerides:

Comprise mono-, di-, and triglycerides.

Microorganisms

Microorganisms are organisms which are microscopic, making them toosmall to be seen by the unaided human eye. Microorganisms includebacteria, fungi, archaea, protists and viruses. Most micro-organisms aresingle-celled, or unicellular organisms, but there are unicellularprotists that are visible to the human eye, and some multicellularspecies are microscopic. Microorganisms live almost everywhere on earthwhere there is liquid water, including hot springs on the ocean floorand deep inside rocks within the earth's crust. Such habitats are livedin by extremophiles.

In the context of the present invention microorganisms do not includelive yeast.

Lactic Acid Bacteria

(or Lactobacillales) are an order of Gram-positive, low-GC (lowguanine-cytosine content), acid-tolerant, generally nonsporulating,non-respiring, either rod or coccus-shaped bacteria that share commonmetabolic and physiological characteristics. These bacteria, usuallyfound in decomposing plants and milk products, produce lactic acid asthe major metabolic end product of carbohydrate bio-conversion. Thelactic acid bacteria are genera of microorganism which produce organicacids, such as lactic acid and acetic acid, as metabolic products ofcarbohydrate bio-conversion. The genera are in particular, but are notlimited to, Lactobacillus, Pediococcus, Lactococcus, Enterococcus,Weisella, Streptococcus, and Leuconostoc.

Other Genera

In the context of the present invention, other genera refer to the mostrelevant other bacterial genera in relation to the invention. Theycomprise a number of genera which also produce organic acids, such aslactic acid and acetic acid, as metabolic products of carbohydratebio-conversion, but often to a lesser extent than the lactic acidbacteria.

In the context of the present invention other genera than the lacticacid comprises, but are not limited to, Bacillus, Bifidobacterium,Brevibacillus, Propionibacterium, Clostridium, and Geobacillus.

Bacillus are genera in the order of Bacillales. The bacteria aregram-positive, rod-shaped, and form endospores under stressfulconditions. Certain strains are used as probiotics.

Processing Aids:

1. Enzymes

Enzyme(s) is a very large class of protein substances with the abilityto act as catalysts. Commonly, they are divided in six classes, and themain classes falling within the scope of this invention can betransferases that transfer functional groups or hydrolases thathydrolyze various bonds. Typical examples can comprise: protease(s),peptidase(s), (α-)galactosidase(s), amylase(s), glucanase(s),pectinase(s), hemicellulase(s), phytase(s), lipase(s), phospholipase(s),transferase(s), cellulase(s), and oxido-reductase(s).

2. Plant Components and Organic Processing Agents

Some of the functional properties that are important in this contextare: Antioxidant, anti-bacterial action, wetting properties andstimulation of enzyme activity.

The list of plant-based components is huge, but the most important arethe following: Rosemary, thyme, oregano, flavonoids, phenolic acids,saponins, and α- and β-acids from hops e.g. α-lupulic acid for themodulation of soluble carbohydrates.

Furthermore, organic acids e.g. sorbic-, propionic-, lactic-, citric-,and ascorbic acid, and their salts for the adjustment of the pH-value,preservation and chelating properties is part of this group ofprocessing aids.

3. Inorganic Processing Agents

Comprise inorganic compositions for example anticaking and flowimproving agents in the final product e.g. potassium aluminium silicate,etc.

Comprise inorganic acids e.g. hydrochloric acid.

Processed Food Products:

Comprise dairy products, processed meat products, sweets, desserts, icecream desserts, canned products, freeze dried meals, dressings, soups,convenience food, bread, cakes, etc.

Processed Feed Products:

Comprise ready-to-use feed for animals such as piglets, calves, poultry,furred animals, sheep, cats, dogs, fish, and crustaceans, etc.

Pharmaceutical Products:

Comprise products, typically in the form of a tablet or in granulatedform, containing one or more biologically active ingredients intendedfor curing and/or alleviating the symptoms of a disease or a condition.Pharmaceutical products furthermore comprise pharmaceutically acceptableexcipients and/or carriers. The solid bio products herein disclosed arevery well suited for use as a pharmaceutically acceptable ingredient ina tablet or granulate.

Cosmetic Products:

Comprise products intended for personal hygiene as well as improvedappearance such as conditioners and bath preparations.

DETAILED DESCRIPTION OF THE INVENTION

In a first embodiment of the method of the invention at least 20% byweight of the biomass, such as at least 30%, at least 40%, at least 50%,at least 60%, at least 70%, at least 80%, or at least 90% by weight,comprises proteinaceous matter originating from optionally defattedsoya. The soya may also be dehulled.

In a second embodiment of the method of the invention at least 20% byweight of the biomass, such as at least 30%, at least 40%, at least 50%,at least 60%, at least 70%, at least 80%, or at least 90% by weight,comprises proteinaceous matter originating from optionally defatted rapeseeds.

In a third embodiment of the method of the invention the biomasscomprises proteinaceous matter originating from optionally defatted soyain an amount of from 5% to 95% by weight in mixture with proteinaceousmatter originating from optionally defatted rape seed in an amount offrom 95% to 5% by weight optionally in further mixture withproteinaceous matter originating from fava beans, peas, sunflower seedsand/or cereals in amounts to make up a total amount of the proteinaceousmatter of 100% by weight.

In any of the embodiments of the invention the biomass comprisingproteinaceous matter may further comprise oligosaccharides, and/orpolysaccharides, and/or further comprises oils and fats, e.g. from seedsof oil bearing plants.

In any of the embodiments of the invention the solid transformationproduct of the substrate may be a product of the transformation ofcarbohydrates, in particular oligosaccharides and polysaccharides,and/or proteinaceous matter originating from said biomass, such as atransformation product of pulses, such as soya, pea, lupine, sunflower,and/or cereals, such as wheat, or maize, or from seeds of oil bearingplants, e.g. rape seed.

In any of the embodiments of the invention the live microorganism ormixture of live microorganisms may be one or more microorganisms whichcan produce one or more organic compounds, such as organic acids, e.g.formic acid, acetic acid, propionic acid, butyric acid, lactic acid, andsuccinic acid, or alcohols, e.g. ethanol, from carbohydrates.

In any of the embodiments of the invention the live microorganism orcombination of live microorganisms may be one or more organic acidproducing microorganism(s).

In any of the embodiments of the invention the live microorganism orcombination of live microorganisms may be selected from the followinglist of genera:

-   -   Lactobacillus    -   Lactococcus    -   Streptococcus    -   Pediococcus    -   Enterococcus    -   Leuconostoc    -   Weisella    -   Bifidobacterium    -   Bacillus    -   Brevibacillus    -   Propionibacterium    -   Clostridium    -   Trichoderma    -   Candida    -   Aspergillus.

In any of the embodiments of the invention the live microorganism orcombination of live microorganisms may be selected from Lactobacillusstrains, and the mixture may be incubated at a temperature of 15-50° C.

In any of the embodiments of the invention the live microorganism orcombination of live microorganisms may be selected from Lactobacillus,Pediococcus, Enterococcus, Lactococcus, Streptococcus, and Weisellastrains, and the mixture may be incubated at a temperature of 15-50° C.

In any of the embodiments of the invention the live microorganism orcombination of live microorganisms may be selected from Bacillusstrains, and the mixture may be incubated a temperature of 20-60° C.

In any of the embodiments of the invention the live microorganism orcombination of live microorganisms may be selected from Bifidobacteriumstrains, and the mixture may be incubated at a temperature of 20-45° C.

In any of the above embodiments water is added to said substrate ofbiomass in an amount which provides an initial incubation mixture havinga ratio of wet bulk density to dry bulk density from 0.65 to 1.40, suchas 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, 1.00, 1.10, 1.15, 1.20, 1.25,1.30, or 1.35.

In any of the above embodiments the live microorganism or combination oflive microorganisms is used in an amount of 10³ to 10¹¹ CFU (colonyforming units) per g of said substrate of biomass, such as 10³, 10⁴,10⁵, 10⁶, 10⁷, 10⁸, 10⁹, or 10¹⁰ CFU/g substrate of biomass. The skilledperson would now how to select a suitable amount, depending on theselected process conditions, such as reactor dimension, the process timeand temperature, the applied microorganism, and the transformationproduct to be produced.

In any of the embodiments of the invention water is added to thesubstrate in an amount to provide a ratio of wet bulk density to drybulk density from about 0.60 to 1.45 in the substrate, such as fromabout 0.65 to about 1.40, e.g. 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, 1.00,1.10, 1.15, 1.20, 1.25, 1.30, or 1.35.

In any of the embodiments of the invention at least 40% by weight of thebiomass, such as at least 50%, at least 60%, at least 70%, at least 80%or at least 90% by weight, may comprise proteinaceous matter originatingfrom optionally defatted rape seeds, whereas water may be added to thesubstrate in an amount to provide a ratio of wet bulk density to drybulk density from about 0.65 to about 1.10, such as 0.75, 0.80, 0.85,0.90, 0.95, 1.00, or 1.05.

In any of the embodiments of the invention one or more processing aidsselected from enzymes, plant components, and organic and inorganicprocessing agents may be added to the substrate of biomass and/or to theinitial incubation mixture.

In any of the embodiments of the invention the filling degree of saidincubation tank may be kept constant. This will result in a uniformflow.

In any of the embodiments of the invention a processing aid selected asα-galactosidase may be added to the substrate of biomass and/or to theinitial incubation mixture, e.g. an α-galactosidase preparation is addedto the substrate of biomass and/or to the initial incubation mixture inan amount of from 0.05 to 50 α-galactosidase units pr. g. dry matter ofsubstrate of biomass, such as from 0.5 to 25 α-galactosidase units pr.g. dry matter of substrate of biomass, e.g. from 1 to 10, from 2 to 8,from 3 to 6, or from 4 to 5 α-galactosidase units pr. g. dry matter ofsubstrate of biomass.

In any of the embodiments of the invention the incubation can be carriedout under anaerobic conditions. The anaerobic conditions are facilitatedby the present invention.

In any of the embodiments of the invention the water content in theincubation mixture may be from 35% to 70% by weight, such as 40%, 45%,50%, 55%, 60%, or 65% by weight. Thus, the water content in the initialmixture does not exceed 70% by weight and it may vary from e.g. from 40%to 65%, from 45% to 60%, from 48% to 52%, or 50% to 55%, such as 49, 50,51, 52, 53, or 54%.

In any of the embodiments of the invention the mixture is incubated for1-240 hours at 15-70° C. The skilled will know how to optimise thereaction time and the reaction temperature in view of the other reactionconditions, such as the selection of microorganisms. Thus, thetemperature may vary as e.g. 20-65° C., 25-60° C., 30-55° C., 35-50° C.,or 40-45° C.; and the reaction time may be selected as e.g. 2 to 180hours, such as 5 to 150 hours, 7 to 120 hours, 10 to 80 hours, 20 to 60hours, or 28 to 48 hours, at each and every one of the here mentionedtemperature intervals.

In any of the embodiments of the invention the solid transformationproduct of the substrate may by dried, optionally followed by milling.

In any of the embodiments of the invention the substrate mixture may beincubated at a time and a temperature sufficient to inactivate themicroorganisms, anti-nutritional factors and the enzyme(s) if usedpartly or totally, and if desired.

In any of the embodiments of the invention the non-agitated incubationtank may be closed.

In any of the embodiments of the invention the non-agitated incubationtank can be of a vertical, oblong cylindrical or polyhedral type. Theadvantage of using this type is that it is space-saving and as it isnon-agitated the operating costs and maintenance costs for mixingequipment are avoided.

In any of the embodiments of the invention the area in the upper part ofsaid non-agitated incubation tank may be less than the area in the lowerpart i.e. the tank is of conical shape. The advantage of this is thatthe slip effect is increased so that biomasses with a reducedflowability can be used.

In any of the embodiments of the invention the non-agitated incubationtank may have insulating matting or a thermal dimple jacket and means tocontrol the temperature inside the incubation tank.

The solid transformation product of the substrate provided by theinvention may be dried to a water content of not more than 15%, 13%,10%, 6%, 4%, or 2% by weight and optionally be in milled form.

The solid product of the invention can be a product of thetransformation of proteinaceous matter and/or carbohydrates originatingfrom said biomass. The solid transformation product may have reducedcontent of anti-nutritional factors, such as trypsin inhibitors,antigens, flatulence-producing oligosaccharides, e.g. stachyose andraffinose; phytic acid, and lectin.

The solid product of the invention may comprise at least 40%proteinaceous matter by weight of dry matter originating from soya.

The solid product of the invention may comprise at least 40%proteinaceous matter by weight of dry matter originating from rape seed.

The solid product of the invention may comprise proteins in an amount of30-65% by weight on dry matter basis originating from plant parts ofsoya, rape seed, or sun flower, or mixtures thereof.

Finally, the invention provides a food, feed, cosmetic or pharmaceuticalproduct or a nutritional supplement containing from 1% to 99% by weightof a solid transformation product produced according to the invention.

EXAMPLES

Density Ratio

Example 1

Ratio of Wet Bulk Density/Dry Bulk Density for Preferred SubstratesBased on Various Biomasses

1.1 Biomasses Used in the Procedure:

Soya

The soya used was defatted Soya Bean Meal (SBM).

Maize

The maize used was whole maize, ground on a hammer mill through a 3.5 mmsieve.

Wheat

The wheat used was whole wheat, ground on a hammer mill through a 3.5 mmsieve.

Sunflower

The sunflower used was defatted Sunflower Seed Meal (SSM).

Rapeseed

The rapeseed used was defatted Rape Seed Meal (RSM).

Fava Beans

The beans used were whole fava beans.

Pea Protein

The pea protein used was a pea protein concentrate.

1.2 Description of the Procedure:

The amount(s) of biomass and water tabulated in the following was mixedfor ten minutes followed by fifty minutes of equilibration in a closedcontainer.

After this the material was poured into a measuring cup of 500 mL andits mass determined by weighing the cup and subtracting the tare of thecup.

The bulk density was calculated as mass/untapped volume in kg/m³.

The dry bulk density used was the measured bulk density of the biomasswithout addition of water.

The wet bulk density was the bulk density of the biomass with addedwater.

The ratio was calculated as wet bulk density divided by the dry bulkdensity.

The moisture content of the biomasses was determined by drying toconstant weight.

After addition of water the moisture in the mixture was determined bycalculation.

1.3 Results:

The results for 100% soya and 80% mixtures with soya are tabulated inthe following:

Bulk Fava Water Moisture Density Soya Maize Wheat Sunflower Rapeseedbean Pea In g In % kg/m³ Ratio 1000 g  0 10.9 665 — 1000 g  100 19.0 6380.96 1000 g  250 28.7 500 0.75 1000 g  450 38.6 476 0.72 1000 g  75049.1 470 0.71 1000 g  900 53.1 572 0.86 1000 g  1100 57.6 655 0.98 1000g  1400 62.9 715 1.07 1000 g  1900 69.3 889 1.34 800 g 200 g 0 11.4 703— 800 g 200 g 450 38.9 617 0.88 800 g 200 g 900 53.4 634 0.90 800 g 200g 1900 69.4 1008 1.43 800 g 200 g 0 11.7 694 — 800 g 200 g 450 39.1 5800.84 800 g 200 g 900 53.5 623 0.90 800 g 200 g 1900 69.5 960 1.38 800 g200 g 0 10.4 683 — 800 g 200 g 450 38.2 554 0.81 800 g 200 g 900 52.9598 0.88 800 g 200 g 1900 69.1 926 1.36 800 g 200 g 0 11.3 711 — 800 g200 g 100 19.4 576 0.81 800 g 200 g 250 29.0 514 0.72 800 g 200 g 45038.8 483 0.68 800 g 200 g 750 49.3 490 0.69 800 g 200 g 900 53.3 5970.84 800 g 200 g 1100 57.8 528 0.74 800 g 200 g 1900 69.4 908 1.28 800 g200 g 0 11.1 691 — 800 g 200 g 450 38.7 569 0.82 800 g 200 g 900 53.2605 0.88 800 g 200 g 1900 69.3 941 1.36 800 g 200 g 0 11.2 703 — 800 g200 g 450 38.7 488 0.69 800 g 200 g 900 53.2 728 1.04 800 g 200 g 190069.4 964 1.37

The results for 60% and 40% of soya mixtures with maize, sunflower andrapeseed as well as 100% rapeseed are tabulated in the following:

Bulk Moisture Density Soya Maize Sunflower Rapeseed Water In % kg/m³Ratio 600 g 400 g  0 g 11.8 703 — 600 g 400 g 250 g 29.5 651 0.93 600 g400 g 450 g 39.2 626 0.89 600 g 400 g 750 g 49.6 631 0.90 600 g 400 g900 g 53.6 666 0.95 600 g 400 g 1100 g  58.0 723 1.03 600 g 400 g 1400g  63.3 796 1.13 600 g 400 g  0 g 10.0 644 — 600 g 400 g 100 g 18.2 5300.82 600 g 400 g 250 g 28.0 435 0.68 600 g 400 g 450 g 37.9 433 0.67 600g 400 g 750 g 48.6 436 0.68 600 g 400 g 900 g 52.6 480 0.75 600 g 400 g1100 g  57.1 449 0.70 600 g 400 g 1400 g  62.5 616 0.96 600 g 400 g  0 g11.7 643 — 600 g 400 g 100 g 19.7 560 0.82 600 g 400 g 250 g 29.4 5020.78 600 g 400 g 450 g 39.1 503 0.78 600 g 400 g 750 g 49.5 492 0.77 600g 400 g 900 g 53.5 516 0.80 600 g 400 g 1100 g  57.9 545 0.85 600 g 400g 1400 g  63.2 655 1.02 400 g 600 g  0 g 12.3 718 — 400 g 600 g 250 g29.9 636 0.89 400 g 600 g 450 g 39.5 638 0.89 400 g 600 g 750 g 49.9 6660.93 400 g 600 g 900 g 53.8 721 1.00 400 g 600 g 1100 g  58.2 802 1.12400 g 600 g 1400 g  63.5 988 1.38 400 g 600 g  0 g 9.5 654 — 400 g 600 g100 g 17.7 535 0.82 400 g 600 g 250 g 27.6 422 0.65 400 g 600 g 450 g37.6 487 0.74 400 g 600 g 750 g 48.3 491 0.75 400 g 600 g 900 g 52.4 5120.78 400 g 600 g 1100 g  56.9 585 0.89 400 g 600 g 1400 g  62.3 612 0.94400 g 600 g  0 g 12.1 658 — 400 g 600 g 100 g 20.1 556 0.84 400 g 600 g250 g 29.7 471 0.72 400 g 600 g 450 g 39.4 458 0.70 400 g 600 g 750 g49.8 486 0.74 400 g 600 g 900 g 53.7 486 0.74 400 g 600 g 1100 g  58.1531 0.81 400 g 600 g 1400 g  63.4 605 0.92  0 g 1000 g   0 g 12.9 616 — 0 g 1000 g  100 g 20.8 484 0.79  0 g 1000 g  250 g 30.3 438 0.71  0 g1000 g  450 g 39.9 457 0.74  0 g 1000 g  750 g 50.2 507 0.82  0 g 1000g  900 g 54.1 535 0.87  0 g 1000 g  1100 g  58.5 585 0.95  0 g 1000 g 1400 g  63.7 688 1.12

Example 2

Ratio of Wet Bulk Density/Dry Bulk Density for Substrates Based onVarious Biomasses and Used in Experiments with Various Microorgansims

The determination of bulk density was performed by pouring an amount ofmaterial (approx. 250 ml) in a 250 ml measuring cylinder and reading thevolume after leveling the surface by gently shaking the cylinder.Following this, the weight of the material was determined. Dry bulkdensities and wet bulk densities were done in triplicates.

The results are summarised in the following table:

Density ratio = Dry matter in wet bulk density/ Biomass % by weight drybulk density 100% SBM 35 1.13 100% SBM 40 0.95 100% SBM 42.5 0.86 100%SBM 52 0.85 100% SBM 55 0.84 80% SBM + 20% RSM 35 1.05 80% SBM + 20% RSM42.5 0.88 80% SBM + 20% RSM 52 0.78 60% SBM + 40% SSM 35 0.94 60% SBM +40% SSM 42.5 0.84 60% SBM + 40% SSM 52 0.73

Lab-Scale Incubation Tests of New Technology Method

The following examples 3 to 9 were lab scale experiments conducted underthe following conditions:

Background:

The background for the following lab-scale incubation tests was toimitate the conditions in the method of the present invention.

Materials and Methods:

Materials

Biomasses: Soya Bean Meal (SBM), Rape Seed Meal (RSM) and Sunflower SeedMeal (SSM) —as described in section 1.1.

Water: Normal tap water

Microorganisms: The microorganism(s) used are specified for eachexample. For all experiments, unless indicated in the specific example,microorganisms were dosed with approximately 10⁸ CFU/g DM. Lactic acidbacteria and Bifidobacteria were grown in MRS broth, washed in 0.9%NaCl, and dosed to the incubation based on a relationship between OD₆₀₀and CFU/ml. The ml amount needed to dose 10⁸ CFU/g DM was subtractedfrom the total water amount stated under each example. For the Bacillusstrains, most of them were dosed as dry formulated cultures, butGeobacillus denitrificans and Bacillus smithii were grown in NutrientBroth, and washed in the same way, and dosed in the same way, asdescribed for the Lactic acid bacteria strains.

The microorganisms and their origin used in the examples are shown inthe following table:

Strain Origin Lactobacillus plantarum Pangoo Lactobacillus paracasei5622 DSMZ Lactobacillus fermentum Bio Growing Lactobacillus acidophilusBio Growing Lactobacillus delbruckii bulgaricus Bio GrowingLactobacillus debruckii sunkii 24966 DSMZ Lactobacillus farci minis Ownisolate Lactobacillus formosensis Own isolate Lactobacillus salivarius20554 DSMZ Bacillus coagulans Pangoo Bacillus licheniformis BioCatBacillus subtilis BioCat Bacillus smithii 2319 DSMZ Lactococcus lactisBio Growing Bifidobacterium animalis Bio Growing Pediococcusacidolactici Pangoo Enterococcus faecium Pangoo Enterococcus faecalisPangoo Enterococcus durans Own isolate Weisella hellenica Own isolateStreptococcus thermophiles Bio Growing Geobacillus thermodenitrificans466 DSMZ DSMZ: Deutsche Sammlung von Mikroorganismen und Zellkulturen

Processing aid: α-galactosidase from Bio-Cat (12,500 U/g). Theα-galactosidase was dosed in 1 ml water, which was substrated from thetotal addition of water stated in the table of each example.

Experimental Method Used

Incubation Tank:

To imitate bio-conversion conditions where oxygen become non-available,bio-conversion where performed in strong plastic bags, squeezed by handto remove air and closed tightly with a strap, still allowing CO₂ toescape.

Incubation:

Samples were incubated at different temperatures, different watercontents and at different length in time, specified for each example.The incubation was stopped by heating 100° C. for 30 min.

Analytical Methods:

Acid Analysis:

The analysis was conducted by LUFA Oldenburg, Germany, using an aqueousdigestion with membrane filtration and subsequent measurement by an ionchromatograph.

Sucrose and Galactose (Sugars):

The content of sucrose and galactose was determined by thin-layerchromatography.

Stationary phase—Silica gel 60 (Merck 1.05553.0001)

Mobile phase—120 mL n-butanol, 80 mL pyridine and 60 mL demineralizedwater

Spots are visualized with a liquid composed of 8 g diphenylamine, 335 mLacetone 8 mL aniline and 60 mL phosphoric acid.

Sugar concentrations were determined by comparison with known standards.

pH:

pH was measured in 10% DM dilutions with a HQ 411d from HACH.

CFU:

CFU were determined by plate spreading, using MRS agar plates for lacticacid bacteria, and Nutrient agar for the Bacillus strains.

Example 3

Testing Different Production Organisms (LAB) at 20° C., at Different DryMatter Ratios

Experimental Set-Up:

Inoculation SBM Dry matter level (88% DM) α-galactosidase Water Strain %of weight CFU/g DM g mg Ml Lactobacillus 42.5 1*10⁸ 113.6 120 122salivarius Lactobacillus 42.5 1*10⁸ 113.6 120 122 debruckii sunkiiLactobacillus 35 1*10⁸ 113.6 120 172 plantarum Lactobacillus 42.5 1*10⁸113.6 120 122 plantarum Lactobacillus 52 1*10⁸ 113.6 120 79 plantarumLactobacillus 35 1*10⁸ 113.6 120 172 paracasei Lactobacillus 42.5 1*10⁸113.6 120 122 paracasei

Samples were incubated in a 20° C. thermostatic water bath.

Results:

After 44 hours of incubation the following results were obtained,showing growth, sugar conversion and acid production:

Inoculation DM Lactic acid Acetic acid Total acid level SucroseGalactose Strain % % of DM % of DM % of DM pH CFU/g DM % of DM % of DMLactobacillus 35 4.9 1.2 6.1 4.9 3*10¹⁰ 0 0 plantarum Lactobacillus 42.53.7 1.3 5.0 5.2 2*10¹⁰ 0 0 plantarum Lactobacillus 52 3.2 0.9 4.1 5.22*10¹⁰ 0 0 Plantarum

After 116 hours of incubation the following results were obtained,showing growth, sugar conversion and acid production:

Inoculation DM Lactic acid Acetic acid Total acid level SucroseGalactose Strain % % of DM % of DM % of DM pH CFU/g DM % of DM % of DMLactobacillus 42.5 3.4 1.0 4.4 4.9 9.5*10⁹  0.5 1.6 salivariusLactobacillus 42.5 3.7 0.5 4.2 4.9 3.9*10⁹  0 1.6 debruckii sunkiiLactobacillus 35 7.3 1.1 8.4 4.5 2.0*10¹⁰ 0 0 plantarum Lactobacillus42.5 5.7 1.1 6.8 4.7 2.3*10¹⁰ 0 0 plantarum Lactobacillus 52 5.1 1.2 6.34.8 2.2*10¹⁰ 0 0 plantarum Lactobacillus 35 4.7 0.8 5.5 4.8 1.9*10¹⁰ 6 0paracasei Lactobacillus 42.5 3.2 0.6 3.8 4.8 1.8*10¹⁰ 6 0 paracasei Partof the sugars was still bound in oligosaccharides in this experiment,even after 166 hours. The potential for acid production is therebylarger than obtained in this test.

Example 4

Testing Different Production Organisms (LAB) at 30° C., at 40% DM

Experimental Set-Up:

Inoculation SBM Dry matter level (88% DM) α-galactosidase Water Strain %of weight CFU/g DM G mg Ml Lactobacillus 40 1*10⁸ 68.2 72 82 plantarumLactococcus 40 1*10⁸ 68.2 72 82 lactis Enterococcus 40 1*10⁸ 68.2 72 82faecium

Samples were incubated in a 30° C. thermostatic water bath.

Results:

After 45 hours of incubation the following results were obtained,showing growth, sugar conversion and acid production:

Inoculation DM Lactic acid Acetic acid Total acid level SucroseGalactose Strain % % of DM % of DM % of DM pH CFU/g DM % of DM % of DMLactobacillus 40 6.2 1.1 7.3 4.6 1*10¹⁰ 0 1.4 plantarum Lactococcus 403.7 0.9 4.6 4.8 1*10¹⁰ 1.8 1.8 lactis Enterococcus 40 5.1 1.4 6.5 4.82*10¹⁰ 0.4 1.4 faecium

After 69 hours of incubation the following results were obtained,showing sugar conversion and acid production (CFU not determined):

Inoculation DM Lactic acid Acetic acid Total acid level SucroseGalactose Strain % % of DM % of DM % of DM pH CFU/g DM % of DM % of DMLactobacillus 40 7.0 1.0 8.0 4.5 0 0.5 plantarum Lactococcus 40 4.3 1.25.5 4.6 1.8 1.2 lactis Enterococcus 40 5.8 1.3 7.1 4.6 0 0.6 faecium

Example 5

Testing Different Production Organisms at 37° C., at Different DryMatter Ratios

Experimental Set-Up:

Inoculation Exp. Dry matter level SBM α-galactosidase Water Strain No. %of weight CFU/g DM (88% DM) mg Ml Lactobacillus 1 35 1*10⁸ 113.6 120 172plantarum Lactobacillus 2 42.5 1*10⁸ 113.6 120 122 plantarumLactobacillus 3 42.5 1*10⁸ 113.6 Not added 122 plantarum Lactobacillus 442.5 1*10⁷ 68.2 72 73 plantarum Lactobacillus 5 42.5 1*10⁹ 68.2 72 73plantarum Lactobacillus 6 52 1*10⁸ 113.6 120 79 plantarum Lactobacillus7 35 1*10⁸ 113.6 120 172 paracasei Lactobacillus 8 42.5 1*10⁸ 113.6 120122 paracasei Lactobacillus 9 52 1*10⁸ 113.6 120 79 paracasei Bacillus10 35 1*10⁸ 68.2 72 103 coagulans Bacillus 11 42.5 1*10⁸ 68.2 72 73coagulans Bacillus 12 42.5 1*10⁸ 113.6 Not added 122 coagulans Bacillus13 42.5 1*10⁷ 68.2 72 73 coagulans Bacillus 14 55 1*10⁸ 68.2 72 41coagulans Bacillus 15 35 1*10⁸ 68.2 72 103 licheniformis Bacillus 1642.5 1*10⁸ 68.2 72 73 licheniformis Bacillus 17 55 1*10⁸ 68.2 72 41licheniformis Bacillus 18 35 1*10⁸ 68.2 72 103 subtilis Bacillus 19 42.51*10⁸ 113.6 120 122 subtilis Bacillus 20 55 1*10⁸ 68.2 72 41 subtilisLactobacillus 21 42.5 1*10⁸ 68.2 72 73 fermentum Lactobacillus 22 42.56*10⁷ 68.2 72 73 acidophilus Lactobacillus 23 42.5 2*10⁷ 68.2 72 73delbruckii bulgaricus Lactobacillus 24 42.5 6*10⁶ 68.2 72 73 farciminisLactobacillus 25 42.5 1*10⁸ 113.6 120 122 formosensis Lactococcus 2642.5 4*10⁷ 68.2 72 73 lactis Bifidobacterium 27 42.5 1*10⁸ 68.2 72 73animalis Pediococcus 28 42.5 1*10⁸ 68.2 72 73 acidolactici Enterococcus29 42.5 1*10⁸ 68.2 72 73 faecium Enterococcus 30 42.5 1*10⁸ 68.2 72 73faecalis Enterococcus 31 42.5 1*10⁸ 113.6 120 122 durans Weisella 3242.5 1*10⁸ 113.6 120 122 hellenica Lactobacillus 33 42.5 1*10⁸ and 113.6120 122 salivarius + 3*10⁷ Lactobacillus paracasei Streptococcus 34 42.55*10⁷and 113.6 120 122 thermophilus + 5*10⁷ Bifidobacterium animalisPediococcus 35 42.5 5*10⁷and 113.6 120 122 acidolactici + 5*10⁷Lactobacillus plantarum Lactobacillus 36 42.5 5*10⁷and 113.6 120 122farciminis + 5*10⁷ Lactobacillus plantarum Lactobacillus 37 42.5 1*10⁸113.6 120 122 plantarum + sucrose (5% of DM)

Results:

After 18.5 to 20 hours of incubation the following results wereobtained, showing growth, sugar conversion and acid production

Inoculation Exp DM Lactic acid Acetic acid Total acid level CFU/gSucrose Galactose Strain No. % % of DM % of DM % of DM pH DM % of DM %of DM Lactobacillus 1 35 6.1 1.2 7.3 4.6 nd 1 2 plantarum Lactobacillus2 42.5 5.3 1.2 6.5 4.7 nd 1 2 plantarum Inoc: 10⁸ CFU/g Lactobacillus 442.5 5.5 1.2 6.7 5.0 nd 2 2 plantarum Inoc: 10⁷ CFU/g Lactobacillus 542.5 6.5 1.2 7.7 4.8 nd 1 2 plantarum Inoc: 10⁹ CFU/g Lactobacillus 6 524.7 1.1 5.8 4.8 nd 2 2 plantarum Lactobacillus 7 35 5.3 0.1 5.4 4.4 nd 22 paracasei Lactobacillus 8 42.5 4.5 0.1 4.6 4.5 nd 2.6 2 paracaseiBacillus 11 42.5 4.5 1.4 5.9 5.3 8*10⁹ 2.5 2 coagulans Inoc: 10⁸ CFU/gBacillus 13 42.5 3.6 1.4 5.0 5.4 7*10⁹ 2.5 2 coagulans Inoc: 10⁷ CFU/gLactobacillus 24 42.5 4.2 0.1 4.3 4.8 nd 4 3.5 farciminis (no 1)Lactococcus 26 42.5 3.0 2.0 5.0 5.0 nd 2 2 lactis Bifidobacterium 2742.5 4.1 2.0 6.1 5.0 nd 2 2 animalis Pediococcus 28 42.5 3.7 1.5 5.2 5.1nd 2 2 acidolactici Enterococ- 29 42.5 5.4 1.4 6.8 5.1 nd 2 2 cusfaecium Lactobacillus 33 42.5 5.1 0.1 5.2 4.4 nd 2 2 salivarius +Lactobacillus paracasei Streptococcus 34 42.5 4.1 1.9 6.0 4.9 nd 2 2thermophiles + Bifidobacterium animalis Pediococcus 35 42.5 5.4 1.2 6.64.7 nd 1 2 acidolactici + Lactobacillus plantarum Lactobacillus 36 42.56.0 0.9 6.9 4.5 nd 1 2 farciminis + Lactobacillus plantarumLactobacillus 37 42.5 5.3 1.1 6.4 4.7 nd 6 2 plantarum + sucrose nd: notdetermined

After 42.5 and 44 hours of incubation the following results wereobtained, showing growth, sugar conversion and acid production:

Inoculation Exp DM Lactic acid Acetic acid Total acid level CFU/gSucrose Galactose Strain No. % % of DM % of DM % of DM pH DM % of DM %of DM Lactobacillus 1 35 7.3 1.0 8.3 4.4  1*10¹⁰ 0 1.4 plantarumLactobacillus 2 42.5 6.8 1.2 8.0 4.4  1*10¹⁰ 0 1.8 plantarum Inoc: 10⁸CFU/g Lactobacillus 3 42.5 3.6 1.2 4.8 5.1 5*10⁹ 0 0 plantarum (noα-gal) Lactobacillus 4 42.5 7.3 1.5 8.8 4.6 nd 0 1.1 plantarum Inoc: 10⁷CFU/g Lactobacillus 5 42.5 7.9 1.1 9.0 4.6 nd 0 1.25 plantarum Inoc: 10⁹CFU/g Lactobacillus 6 52 6.6 1.2 7.8 4.5  1*10¹⁰ 0 1.8 plantarumLactobacillus 7 35 7.2 0.1 7.3 4.1  3*10¹⁰ 0 2.4 paracasei Lactobacillus8 42.5 6.6 0.1 6.7 4.2  2*10¹⁰ 0.5 2.6 paracasei Lactobacillus 9 52 5.30.1 5.4 4.4  2*10¹⁰ 3 3 paracasei Bacillus 10 35 6.9 1.2 8.1 4.5 nd 01.3 coagulans Bacillus 11 42.5 8.2 1.3 9.5 4.6 4*10⁹ 0.4 1 coagulansInoc: 10⁸ CFU/g Bacillus 12 42.5 5.5 1.2 6.7 4.7 2*10⁹ 0 0 coagulans (noα-gal) Bacillus 13 42.5 7.3 1.3 8.6 4.7 3*10⁹ 0.5 1 coagulans Inoc: 10⁷CFU/g Bacillus 14 55 3.7 0.8 4.5 5.1 nd 2 2 coagulans Bacillus 15 35 2.70.0 2.7 5.1 nd 2.5 4 licheniformis Bacillus 16 42.5 0.8 0.0 0.8 6.0 nd2.5 4 licheniformis Bacillus 17 55 0.2 0.0 0.2 6.4 nd 2.7 3.3licheniformis Bacillus 18 35 2.4 0.1 2.5 5.1 nd 3 4 subtilis Bacillus 1942.5 2.5 0.9 3.4 5.3 3*10⁹ 3.6 2.6 subtilis Bacillus 20 55 0.5 0.1 0.66.0 nd 5 2 subtilis Lactobacillus 21 42.5 4.6 2.1 6.7 4.9  5*10¹⁰ 1 1fermentum Lactobacillus 22 42.5 4.2 0.2 4.4 4.7 4*10⁹ 1 1 acidophilusLactobacillus 23 42.5 3.7 1.7 5.4 5.2 8*10⁹ 3.3 2.5 delbruckiibulgaricus Lactobacillus 24 42.5 7.9 0.3 8.2 4.2 5*10⁹ 0.8 2.8farciminis Lactobacillus 25 42.5 6.5 0.2 6.7 4.2 3*10⁹ 0.5 2 formosensisLactococcus 26 42.5 4.0 2.3 6.3 4.8 8*10⁹ 0.8 1 lactis Bifidobacterium27 42.5 4.5 2.1 6.6 4.9 6*10⁹ 1 0.8 animalis Pediococcus 28 42.5 6.9 1.58.4 4.6 9*10⁹ 0.5 0.7 acidolactici Enterococcus 29 42.5 7.6 1.5 9.1 4.67*10⁹ 0.5 0.7 faecium Enterococcus 30 42.5 5.8 1.5 7.3 4.7 9*10⁹ 0.3 0.3faecalis Enterococcus 31 42.5 2.7 0/2 2.9 4.9 2*10⁹ 3 2 durans Weisella32 42.5 4.1 1.6 5.7 4.9 3*10⁹ 1 1 hellenica Lactobacillus 33 42.5 6.20.1 6.3 4.2  1*10¹⁰ 1 1.9 salivarius + Lactobacillus paracaseiStreptococcus 34 42.5 5.1 2.0 7.1 4.8  8*10¹⁰ 0.2 1 thermosphiles +Bifidobacterium animalis Pediococcus 35 42.5 6.9 1.2 8.1 4.4  1*10¹⁰ 0 1acidolactici + Lactobacillus plantarum Lactobacillus 36 42.5 7.4 1.0 8.44.4 9*10⁹ 0 1.4 farciminis + Lactobacillus plantarum Lactobacillus 3742.5 6.5 1.0 7.5 4.4  1*10¹⁰ 4 2.2 plantarum + sucrose nd: notdetermined

After 116 hours of incubation the following results were obtained,showing sugar conversion and acid production.

Exp. DM Lactic acid Acetic acid Total acid Sucrose Galactose Strain No.% % of DM % of DM % of DM pH % of DM % of DM Bacillus 10 35 7.7 1.2 8.94.3 0 0 coagulans Bacillus 11 42.5 7.7 1.2 8.9 4.3 0.1 0.5 coagulansBacillus 14 55 4.8 0.7 5.5 4.8 1.2 1.6 coagulans Bacillus 15 35 2.5 0.12.6 4.8 0.3 3.5 licheniformis Bacillus 16 42.5 1.7 0.1 1.8 5.5 2 3.3licheniformis Bacillus 17 55 0.4 0.1 0.5 6.3 2.9 3.3 licheniformisBacillus 18 35 1.6 0.1 1.7 4.9 0 3.5 subtilis Bacillus 19 42.5 1.4 0.11.5 5.1 1.5 3.5 subtilis Bacillus 20 55 0.8 0.2 1.0 5.9 5 3 subtilis

Example 6

Testing Different Production Organisms at 44° C., at 40% DM

Experimental Set-Up:

Inoculation SBM Dry matter level 88% DM α-galactosidase Water Strain %of weight CFU/g DM g mg Ml Lactobacillus 40 1*10⁸ 68.2 72 73 plantarumPediococcus 40 1*10⁸ 68.2 72 73 acidolactici Bacillus 40 1*10⁸ 68.2 7273 coagulans Bacillus 40 1*10⁸ 68.2 72 73 licheniformis Bacillussubtilis 40 1*10⁸ 68.2 72 73

Samples were incubated in a 44° C. thermostatic water bath.

Results:

After 20 hours of incubation the following results were obtained,showing growth, sugar conversion and acid production:

Inoculation DM Lactic acid Acetic acid Total acid level SucroseGalactose Strain % % of DM % of DM % of DM pH CFU/g DM % of DM % of DMLactobacillus 40 5.1 0.2 5.3 4.8 Nd 3 2.5 plantarum Pediococcus 40 4.70.2 4.9 4.8 Nd 3 3 acidolactici Bacillus 40 4.4 0.1 4.5 5.0  2*10¹⁰ 22.5 coagulans Bacillus 40 1.1 0.0 1.1 6.0 2*10⁸ 2.5 3.6 licheniformisBacillus 40 0.7 0.2 0.9 6.0 1*10⁹ 6.5 3.8 subtilis nd: not determined

After 44 hours of incubation the following results were obtained,showing sugar conversion and acid production (CFU not determined):

DM Lactic acid Acetic acid Total acid Sucrose Galactose Strain % % of DM% of DM % of DM pH % of DM % of DM Lactobacillus 40 6.8 0.3 7.1 4.4 32.2 plantarum Pediococcus 40 6.6 0.3 6.9 4.4 2 1.5 acidolactici Bacillus40 7.2 0.2 7.4 4.4 0.5 1.0 coagulans Bacillus 40 1.5 0.1 1.6 5.9 1 2.9licheniformis Bacillus 40 1.3 0.1 1.4 5.7 4.5 2.9 subtilis

Example 7

Testing Different Production Organisms at 52° C., at 52% DM

Experimental Set-Up:

Inoculation SBM Dry matter level (88% DM) α-galactosidase Water Strain %of weight CFU/g DM g mg Ml Bacillus smithii 42.5 1*10⁸ 113.6 120 172Bacillus smithii 42.5 1*10⁸ 113.6 No addition 172 Bacillus 42.5 1*10⁸113.6 120 172 licheniformis Bacillus 42.5 1*10⁸ 113.6 No addition 172licheniformis Bacillus 42.5 1*10⁸ 113.6 120 172 coagulans

Samples were incubated in a 52° C. thermostatic water bath.

Results:

After 116.5 hours of incubation the following results were obtained,showing growth, sugar conversion and acid production

Inoculation DM Lactic acid Acetic acid Total acid level SucroseGalactose Strain % % of DM % of DM % of DM pH CFU/g DM % of DM % of DMBacillus smithii 42.5 3.3 0.1 3.4 5.2 1*10⁶ 4 2 Bacillus smithii 42.52.2 0.1 2.3 5.3 Nd 2 0 (no α-gal) Bacillus 42.5 3.8 0 3.8 5.4 5*10⁷ 3 2licheniformis Bacillus 42.5 2.7 0 2.7 5.5 Nd 0.5 0 licheniformis (noα-gal) Bacillus 42.5 1.8 0.2 2.0 4.9 4*10⁸ 1.5 1.5 coagulans nd: notdetermined

Example 8

Testing Different Production Organisms at 60° C.

Experimental Set-Up:

Inoculation SBM Dry matter level (88% DM) α-galactosidase Water Strain %of weight CFU/g DM g mg Ml Bacillus coagulans 42.5 1*10⁸ 113.6 120 122Bacillus smithii 42.5 1*10⁸ 113.6 120 122 Geobacillus 35 1*10⁸ 113.6 120172 thermodenitrificans

Samples were incubated in a 60° C. incubator.

Results:

After 44.5 and 116.5 hours of incubation the following results wereobtained, showing growth, sugar conversion and acid production

Incubation Inoculation Time DM Lactic acid Acetic acid Total acids levelSucrose Galactose Strain Hours % % of DM % of DM % of DM pH CFU/g DM %of DM % of DM Bacillus 116.5 42.5 1.3 0.2 1.5 5.3 6*10⁶ 7 4 coagulansBacillus 116.5 42.5 0.8 0.4 1.2 5.7 5*10⁶ 7 4 smithii Geobacillus 44.535 2.0 0.2 2.2 5.2 9*10⁷ 8 4 thermodenitrificans

Example 9

Bioconversion with Alternative Biomasses

Incubation at 37° C. for 42.5 to 45.5 hours

Inoculation SBM RSM SSM Dry matter level (88% DM) (88% DM) (91% DM)α-galactosedase Water Strain % of weight CFU/g DM g g g mg MlLactobacillus 35 1*10⁸ 113.6 — — 120 172 plantarum Lactobacillus 42.51*10⁸ 113.6 — — 120 122 plantarum Lactobacillus 52 1*10⁸ 113.6 — — 12079 plantarum Lactobacillus 35 1*10⁸ 90.1 22.8 — 120 172 plantarumLactobacillus 42.5 1*10⁸ 90.1 22.8 — 120 122 plantarum Lactobacillus 521*10⁸ 90.1 22.8 — 120 79 plantarum Lactobacillus 35 1*10⁸ 67.8 — 43.8120 174 plantarum Lactobacillus 42.5 1*10⁸ 67.8 — 43.8 120 124 plantarumLactobacillus 52 1*10⁸ 67.8 — 43.8 120 81 plantarum Bacillus 35 1*10⁸68.2 — — 72 103 coagulans Bacillus 42.5 1*10⁸ 113.6 — — 120 122coagulans Bacillus 55 1*10⁸ 68.2 — — 72 41 coagulans Bacillus 35 1*10⁸90.1 22.8 — 120 172 coagulans Bacillus 42.5 1*10⁸ 90.1 22.8 — 120 122coagulans Bacillus 52 1*10⁸ 90.1 22.8 — 120 79 coagulans Bacillus 351*10⁸ 67.8 — 43.8 120 174 coagulans Bacillus 42.5 1*10⁸ 67.8 — 43.8 120124 coagulans Bacillus 52 1*10⁸ 67.8 — 43.8 120 81 coagulans

Results:

After 42.5 to 45.5 hours of incubation the following results wereobtained, showing growth, sugar conversion and acid production

Inoculation DM Bio- Lactic acid Acetic acid Total acids level SucroseGalactose Strain % mass % of DM % of DM % of DM pH CFU/g DM % of DM % ofDM Lactobacillus 35 SBM 7.2 0.1 7.3 4.1  3*10¹⁰ 0 2.4 plantarumLactobacillus 42.5 SBM 6.8 1.2 8.0 4.4  1*10¹⁰ 0 1.8 plantarumLactobacillus 52 SBM 6.6 1.2 7.8 4.5  1*10¹⁰ 0 1.8 plantarumLactobacillus 35 SBM/ 7.3 0.9 8.2 4.4 5*10⁹ 0 1 plantarum RSMLactobacillus 42.5 SBM/ 6.8 1.0 7.8 4.4 5*10⁹ 0 1 plantarum RSMLactobacillus 52 SBM/ 5.9 0.9 6.8 4.5 7*10⁹ 0.8 1 plantarum RSMLactobacillus 35 SBM/ 6.5 0.7 7.2 4.4 4*10⁹ 0 1 plantarum SSMLactobacillus 42.5 SBM/ 6.3 0.7 7.0 4.4 4*10⁹ 0 1 plantarum SSMLactobacillus 52 SBM/ 5.7 0.7 6.4 4.4 4*10⁹ 0.8 1 plantarum SSM Bacillus35 SBM 6.9 1.2 8.1 4.5 nd 0 1.3 coagulans Bacillus 42.5 SBM 6.5 1.3 7.84.5 7*10⁹ 0 0.5 coagulans Bacillus 55 SBM 3.7 0.8 4.5 5.1 nd 2 2coagulans Bacillus 35 SBM/ 7.2 0.9 8.1 4.4 2*10⁹ 0 0.5 coagulans RSMBacillus 42.5 SBM/ 6.3 1.0 7.3 4.4 3*10⁹ 0.3 0.8 coagulans RSM Bacillus52 SBM/ 5.6 0.9 6.5 4.5 2*10⁹ 1 1 coagulans RSM Bacillus 35 SBM/ 6.1 0.76.8 4.4 2*10⁹ 0 0 coagulans SSM Bacillus 42.5 SBM/ 5.8 0.7 6.5 4.4 5*10⁹0.3 0.5 coagulans SSM Bacillus 52 SBM/ 4.7 0.7 5.4 4.6 3*10⁹ 1 1coagulans SSM

Example 10

Pilot Scale Bioconversion

Incubator:

The incubator was a pilot scale vertical reactor with a total volume of2.0 m². The incubator was equipped with a temperature probe at the inletas well as at the outlet.

Incubation Mixture:

The incubator was incubated with a preheated mixture of 250 kg soya beanmeal (88% DM); 264 g α-galactosidase from Bio-Cat (12,500 U/g), dryformulation of Bacillus coagulans to reach a final inoculation level of1*10⁷ cells/g DM, and 268 litre tap water. The ratio wet bulkdensity/dry bulk density of the incubation mixture was 0.88. Thisresulted in a DM of 42.5% of the incubation mixture.

Test Procedure:

After filling of the reactor, it was flushed with N₂ gas, to get rid ofO₂. The biomass was incubated at 60 hours at 37° C.

Results:

After 60 hours a product comprising 7.5% of DM lactic acid and 1.3% ofDM acetic acid was obtained.

pH had dropped to 4.6.

Example 11

Large Scale Bioconversion

Incubator:

The reactor used was a vertical cylinder with an effective height of 7.3m and a diameter of 4.3 m.

In the top of the vertical reactor, the feed mixture falls on positionnear the centre of the reactor. For even distribution, a scraper bladeor level arm distributes the inlet feed mixture over the perimeter ofreactor.

In the bottom of the reactor, the product was extracted by means toachieve a uniform residence time for any particle spread on the top ofthe reactor.

Testing Uniform Plug Flow

The inlet and outlet means of the reactor were adjusted to achieve anexpected residence time of 12 hours. For proving the uniformdistribution time, an inert tracer substance was added to the feedmixture. The feed mixture used in the experiment had a natural contentof iron of around 143 mg/kg dry matter (=off-set concentration);therefore, iron sulphate (FeSO₄) was used as a tracer in a concentrationof 1167 mg FeSO₄/kg feed mixture dry matter equal to a total ironcontent of 572 mg Fe/kg total dry matter. At time 0 hours, FeSO₄ wasadded to the feed mixture dosed to the reactor for a period of 60minutes. Samples were drawn every 20 minutes, dried, and analysed forcontent of iron, and it was found that the FeSO₄ enriched product leavesthe reactor 12-13 hours after dosing FeSO₄ to the inlet feed mixture,and a maximum concentration of 355 mg/kg Fe was found at 12.5 hoursafter start.

The invention claimed is:
 1. A method for producing a solidtransformation product of a biomass substrate, wherein the solidtransformation product is a product of the transformation of one or moreof proteinaceous matter and carbohydrates originating from a biomasssubstrate, the method comprising: (a) preparing a substrate of a biomasscomprising carbohydrates and proteinaceous matter that originate fromsoya bean seed, rape seed, or mixtures thereof, wherein at least 20% byweight of said biomass comprises carbohydrates and proteinaceous matteroriginating from soya bean seeds, rape seeds, or mixtures thereof,optionally in further mixture with carbohydrates and proteinaceousmatter originating from one or more of seeds of fava beans, seeds ofpeas, sunflower seeds, seeds of lupine, cereals, and grasses; (b) mixingsaid substrate with a live microorganism or a combination of livemicroorganisms, which live microorganism or combination of livemicroorganisms is not, and does not comprise, live yeast, and addingwater in an amount which provides an initial incubation mixture having awater content from 30% to 70% by weight, and a ratio of wet bulk densityto dry bulk density from 0.60 to 1.45; (c) incubating said initialincubation mixture for 1-240 hours at a temperature of 15-70° C., and(d) recovering solid transformation product from the incubated mixture;wherein the incubating step is performed as a continuous plug-flowprocess in a vertical, non-agitated incubation tank with an inlet forsaid mixture and additives and an outlet for said solid transformationproduct, and wherein transport of the biomass is mediated bygravitational force.
 2. The method according to claim 1, furthercomprising pre-treating said substrate before mixing with said livemicroorganism or said combination of live microorganisms by one or moreselected from disintegration, milling, flaking, heat treatment, pressuretreatment, ultrasonic treatment, hydrothermal treatment, acid treatmentand alkaline treatment.
 3. The method according to claim 1, wherein atleast 30% by weight of said biomass comprises carbohydrates andproteinaceous matter originating from one or more of optionally defattedand optionally dehulled soya bean seeds, optionally defatted rape seeds,and mixtures thereof.
 4. The method according to claim 3, wherein theweight of said biomass comprising carbohydrates and proteinaceous matteroriginating from optionally defatted and/or optionally dehulled soyabean seeds, optionally defatted rape seeds, or mixtures thereof isselected from at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%, and at least 90% by weight of said biomass.
 5. The methodaccording to claim 1, wherein said biomass comprises one or more ofoligosaccharides and polysaccharides, and optionally further comprisesoils and fats.
 6. The method according to claim 1, wherein said solidtransformation product is a product of the transformation ofproteinaceous matter, or of the transformation of carbohydrates, or ofthe transformation of proteinaceous matter and carbohydrates originatingfrom seeds of soya, pea, lupine, or sunflower, or from wheat, maize, orrape seed.
 7. The method according to claim 1, wherein the livemicroorganism or combination of live microorganisms is one or moremicroorganisms which can produce one or more organic acids fromcarbohydrates selected from formic acid, acetic acid, propionic acid,butyric acid, lactic acid, and succinic acid.
 8. The method according toclaim 1, wherein the live microorganism or combination of livemicroorganisms is one or more microorganisms which can produce one ormore alcohols from carbohydrates.
 9. The method according to claim 1,wherein the live microorganism or combination of live microorganisms isof a genus selected from: Lactobacillus Lactococcus StreptococcusPediococcus Enterococcus Leuconostoc Weisella Bifidobacterium BacillusBrevibacillus Propionibacterium Clostridium Trichoderma and Aspergillus.10. The method according to claim 1, wherein the live microorganism orcombination of live microorganisms is selected from one or more ofLactobacillus, Pediococcus, Enterococcus, Lactococcus, Streptococcus,and Weisella strains, and wherein the initial incubation mixture isincubated at a temperature of 15-50° C.
 11. The method according toclaim 1, wherein the live microorganism or combination of livemicroorganisms is selected from Bacillus strains, and wherein theinitial incubation mixture is incubated a temperature of 20-60° C. 12.The method according to claim 1, wherein the live microorganism orcombination of live microorganisms is selected from Bifidobacteriumstrains, and wherein the initial incubation mixture is incubated at atemperature of 20-45° C.
 13. The method according to claim 1, whereinsaid initial incubation mixture is incubated for 2 to 180 hours.
 14. Themethod according to claim 1, wherein water is added to said substrate inan amount which provides an initial incubation mixture having a ratio ofwet bulk density to dry bulk density from 0.65 to 1.40.
 15. The methodaccording to claim 1, wherein water is added to said substrate ofbiomass in an amount which provides an initial incubation mixture havinga ratio of wet bulk density to dry bulk density selected from 0.70,0.75, 0.80, 0.85, 0.90, 0.95, 1.00, 1.10, 1.15, 1.20, 1.25, 1.30, and1.35.
 16. The method according to claim 1, wherein the water content insaid initial incubation mixture is from 35% to 70% by weight.
 17. Themethod according to claim 1, wherein the water content in said initialincubation mixture is selected from 40%, 45%, 50%, 55%, 60%, and 65%.18. The method according to claim 1, wherein said live microorganism orcombination of live microorganisms is used in an amount of 10³ to 10¹¹CFU (colony forming units) per g of said substrate.
 19. The methodaccording to claim 1, further comprising adding one or more processingaids selected from enzymes, plant components, and organic and inorganicprocessing agents to one or more of the substrate and the initialincubation mixture.
 20. The method according to claim 1, furthercomprising adding α-galactosidase to one or more of the substrate andthe initial incubation mixture.
 21. The method according to claim 1,further comprising adding an α-galactosidase preparation to one or moreof the substrate and the initial incubation mixture in an amount of from0.05 to 50 α-galactosidase units per g dry matter of the substrate. 22.The method according to claim 1, wherein the vertical, non-agitatedincubation tank is closed.
 23. The method according to claim 1, whereinsaid incubation is carried out under anaerobic conditions.
 24. Themethod according to claim 1, wherein said non-agitated incubation tankis of a vertical, oblong cylindrical or polyhedral type.
 25. The methodaccording to claim 1, wherein the area in the upper part of saidnon-agitated incubation tank is less than the area in the lower part.26. The method according to claim 1, where said non-agitated incubationtank has insulating matting or a thermal dimple jacket.
 27. The methodaccording to claim 1, wherein the filling degree of said incubation tankis kept constant.